In the context of this disclosure one or more of the following terms may be used.
Promoter analysis in plants can provide information on both the strength of the promoter and its regulation in different tissues. Promoter analysis studies have been performed with either stably-transformed tissues or using transient expression analyses. For stable transformation, the time required for generation of a transgenic plant can be as short as six weeks (An et al. 1986), but can often extend beyond five months when transformation and plant recovery is slow (Santarém and Finer 1999). Production of stably-transformed plants is necessary for a detailed examination of promoter expression; however, quantification of promoter strength and comparative analyses in stably transformed plants can still be difficult due to variation in transgene expression among transgenic clones (Finnegan and McElroy 1994).
Rapid, quantifiable, and reproducible promoter analyses are simplified using transient expression, where gene expression can be observed in as little as 1.5 hours post introduction (Ponappa et al. 1999) and gene expression will not be influenced by copy number or site of integration. Transient expression analysis can be performed via direct DNA introduction into protoplasts using electroporation (Christensen et al. 1992) or PEG (Hartmann et al. 1998), or particle bombardment-mediated transformation into intact plant tissues (Rolfe and Tobin 1991). Agroinfiltration of tobacco (Bendahmane et al. 1999; Vaucheret, 1994) is also commonly used for rapid analysis of transgene effects. Reporter genes such as luciferase (Ow et al, 1986), β-glucuronidase (Samac et al. 2004; Vain et al. 1996) and chloramphenicol acetyl transferase (Kang et al. 2003) are most commonly utilized for quantification of promoter activity. Unfortunately, visualization of luciferase and β-glucuronidase activity requires the addition of an artificial substrate and quantification of promoter activity using all of these reporter genes and requires the extraction of protein from the sample, destroying the sample and eliminating the ability to follow gene expression in the same piece of tissue over time.
The green fluorescent protein (gfp) gene offers tremendous opportunities for promoter analysis in plants since its expression can be followed in the same piece of tissue over extended periods of time (Piston et al. 1999). Although GFP expression has been used in studies to characterize promoter activity (Abebe et al. 2006), reports on the quantification of GFP expression using image analysis (Nagatani et al. 1997), spectofluorometry (Richards et al. 2003) or fluorescence spectroscopy (Stewart et al. 2005) are minimal.
Standard methods are needed for the evaluation of promoter strength based on GFP detection. Recently, an automated robotics system was developed for monitoring GFP expression over time in multiple pieces of tissue (Buenrostro-Nava et al. 2005). The robotics system consisted of a 2-dimensional robotics platform, a cooled CCD camera, and a dissecting fluorescence microscope, all under computer control. Although the monitoring system was initially used for automated image collection of GFP expression in stably-transformed somatic embryos (Buenrostro-Nava et al. 2006) and Agrobacterium (Buenrostro-Nava et al. 2003), it also has utility for rapid quantification of promoter strength using transient expression analyses.
Soybean (Glycine max (L.) Merr.), a valuable agronomic crop world-wide, has the highest transgenic acreage of any crop. As efforts move forward to produce new and improved transgenic soybean, the need for different types of native soybean promoters will continue to increase. Some soybean promoters have already been identified but these promoters direct expression in a tissue-specific (Chen et al. 1986) or inducible manner (Czarnecka et al. 1989; Liu et al. 1994). A strong, constitutive, native soybean promoter, which could replace the constitutive Cauliflower Mosaic Virus 35S (CaMV35S) was sought. Of the strong constitutive plant promoters that have been used extensively for directing transgene expression, the polyubiquitin promoters have received the most widespread attention (Christensen and Quail 1996). A common feature of polyubiquitin promoters is the presence of a leading intron, which is considered part of the promoter, and can influence transgene expression (Christensen and Quail 1996). Removal of the intron from the promoter region either reduces the strength of the promoter (Plesse et al. 2001) or results in complete loss of promoter activity (Wang and Oard 2003).
In addition to a strong constitutive promoter, a developmentally regulated promoter would be useful for comparative studies. Although soybean promoters active during late stages of seed development are available (Chen et al. 1986), EST data from induced soybean somatic embryos (Thibaud-Nissen et al. 2003) now permits the identification of useful early embryo-specific promoters.